The long-term goal of this project is to develop viral vectors that can be used as gene therapy vehicles to treat Parkinson's disease (PD), as well as other chronic neurodegenerative diseases. Such vectors require the incorporation of a promoter element that permits effective and safe regulation of the therapeutic gene through peripheral drug administration. The proposed experiments will focus on tetracycline(tet)-regulated promoter systems in the context of recombinant adeno-associated viral vectors (AAV) and tentiviral vectors which are based on the human immunodeficiency virus (HIV). A set of AAV and HIV viral vectors will be made that incorporate cellular marker and therapeutic genes driven by tet-regulated promoters that can be turned-on or turned-off by administration of the tet analog doxycycline (Dox). Cellular marker genes including humanized green fluorescent protein (hrGFP) and an non-immunogenic gene, rat alkaline phosphatase (rAP), will be used to assess the efficiency and suitability of the vector designs. Quantitative assays including flow cytometry, real-time, quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR), ELISA and computerized morphometry will be applied to assess and compare vectors in cell culture and in the rat nigrostriatal system. In vivo studies will be clone in both normal intact rat brain and in the 6-OHDA progressively lesioned rat model of PD to determine whether damage related to PD will affect the efficiency of vectors containing regulated promoters. These studies will also determine to what extent the therapeutic effects of glial cell line-derived neurotrophic factor (GDNF) gene therapy in this rat model of PD are reversible. Effects of GDNF gene delivery using tet-regulated vectors on dopamine neurons will be evaluated using quantitative morphometric, molecular and behavioral evaluations. All studies will involve assessment of host immune reactions and chromosomal effects of the vectors in collaboration with the Lowenstein and Federoff labs in this consortium. The successful generation of a viral vector that fulfills the requirements of tight regulation, long-term expression and regulatability with minimal host immune responses in the rat CNS will be advanced to non-human primate preclinical trials for PD.